Exonuclease III (Exo III)

Description Exonuclease III (Exo III) exhibits four different catalytic activities (1): PRODUCT INFORMATION  3'5' exodeoxyribonuclease activity specific for double- stranded DNA: Exonuclease III Exo III degrades dsDNA from blunt ends, 5'-overhangs or nicks, releasing 5'-mononucleotides from the (Exo III) 3'-ends of DNA strands and producing stretches of single-stranded DNA. It is not active on 3'-overhang ends Pub. No. MAN0011997 of DNA that are at least four bases long and do not carry Rev. Date 15.July.2016 (Rev. B.00) a 3'-terminal C-residue (2); on single-stranded DNA, or on phosphorothioate-linked nucleotides. #_  3'-phosphatase activity: Lot: _ Expiry Date: _ Exo III removes the 3'-terminal phosphate and generates a 3'-OH group.

 RNase H activity: Exo III exonucleolytically degrades the RNA strand in Components #EN0191 DNA-RNA hybrids.  Apurinic/apyrimidinic- endonuclease activity: Exonuclease III, 200 U/µL 4000 U Exo III cleaves phosphodiester bonds at apurinic or apyrimidinic sites to produce 5'-termini that are base-free 10X Reaction Buffer 0.2 mL deoxyribose 5'-phosphate residues.

Store at -20 °C

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For Research Use Only. Not for use in diagnostic procedures. Applications Note  Creation of unidirectional deletions in DNA fragments in  The rate of DNA digestion by Exo III depends upon conjunction with S1 Nuclease (2, 3), see protocol on back page. temperature, salt concentration and the molar ratio of  Generation of a single-stranded template for dideoxy DNA to enzyme in a reaction mixture (4, 8). Optimal sequencing of DNA (4). reaction conditions should be determined experimentally.  Site-directed mutagenesis (5).  Activity in Thermo Scientific REase Buffers, %  Cloning of PCR products (6). (in comparison to activity in assay buffer)  Preparation of strand-specific probes. Buffers Activity, % Source Thermo Scientific FastDigest, FastDigest™ E.coli cells carrying a cloned E.coli xth gene. Green, O, R, 2X Thermo Scientific Tango, Molecular Weight BamHI, EcoRI 0-25 31 kDa monomer. G, 1X Tango™ 25-50 Definition of Activity Unit B, Ecl136II, KpnI, PacI, SacI 100 One unit of the enzyme catalyzes the release of 1 nmol of acid soluble reaction products from E.coli [3H]-DNA in CERTIFICATE OF ANALYSIS 30 min at 37 °C. Enzyme activity is assayed in the following mixture: Endodeoxyribonuclease Assay 50 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 1 mM DTT and No detectable degradation was observed after incubation 0.05 mM sonicated E.coli [3H]-DNA. of supercoiled plasmid DNA with Exonuclease III. Storage Buffer The enzyme is supplied in: 50 mM Tris-HCl (pH 8.0), Quality authorized by: Jurgita Zilinskiene 50 mM KCl, 1 mM DTT and 50% (v/v) glycerol. 10X Reaction Buffer

660 mM Tris-HCl (pH 8.0 at 30 °C), 6.6 mM MgCl2. Inhibition and Inactivation  Inhibitors: metal chelators, p-chloromercuri benzoate

(50-90% inhibitory at 0.1 mM) (7). (continued on back page)  Inactivated by heating at 70 °C for 10 min. Protocol for Generation of Unidirectional Deletions 6. Select incubation temperature and time as follows: in DNA Fragments (for 25 time points) Temperature, °C 25 30 37 45 Digestion rate, bp/min 80 210 420 600 1. Digest 5-10 µg of DNA with a pair of restriction Mononucleotides are released at base-dependent rates enzymes; one which generates a blunt or in the order: C>A=T>G. DNA termini of different are 5'-overhanging end adjacent to the target sequence, and degraded at different rates, so these guidelines should another which produces a resistant 4 base only be used as an approximation. 3'-overhang close to the priming site. 7. Warm the DNA solution to the digestion temperature. 2. Extract the reaction mixture with 1 volume of Add 500 units Exo III and mix rapidly. Remove 2 µL phenol:chloroform (1:1) and then with 1 volume of aliquots at the chosen time intervals and add to the chloroform:isoamyl alcohol (24:1). Precipitate DNA by tubes containing the cold S1 Nuclease mix. adding 0.1 volume of NaCl/glycogen solution (1.1 M NaCl, 0.25 mg/mL glycogen) and 2 volumes of 8. After all samples have been taken, move the tubes to 95% ethanol. Mix and centrifuge at 10,000 rpm for room temperature for 30 minutes. Add 1 µL S1 "STOP" 10 minutes. Wash the pellet with 1 mL of 70% ethanol. solution (300 mM Tris base, 50 mM EDTA) and heat at Dry the pellet. 70 °C for 10 minutes to inactivate S1 Nuclease. 3. Dissolve DNA in 50 µL of 1X Exo III reaction buffer. 9. Use 7 µL of each sample for gel electrophoresis.

4. Prepare mixture (S1 Nuclease mix) by adding the following: 5X Reaction Buffer for S1 Nuclease* 40 µL S1 Nuclease (#EN0321) 0.5 µL (50 U) Water, nuclease-free (#R0581) to 200 µL * 200 mM sodium acetate (pH 4.5 at 25 °C), 1.5 M NaCl and 10 mM ZnSO4) 5. Add 7.5 µL aliquots of the S1 Nuclease mix into each of 25 numbered tubes, place on ice. References 1. Rogers, S.G. and Weiss, B., Exonuclease III of Escherichia coli K-12, an AP endonuclease, Methods Enzymol., 65, 201-211, 1980. 2. Hoheisel, J.D., On the Activities of Escherichia coli Exonuclease III, Anal.Biochem., 209, 238-246, 1993. 3. Henikoff, S., Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing, Gene, LIMITED USE LABEL LICENSE: Internal Research and Development Use Only. 28, 351-359, 1984. The purchase of this product conveys to the buyer the limited, non-exclusive, non- 4. Guo, Li-He., Wu, R., New rapid methods for DNA transferable right (without the right to resell, repackage, or further sublicense) to use sequencing based on exonuclease III digestion followed this product for internal research and development purposes. No other license is granted to the buyer whether expressly, by implication, by estoppel or otherwise. In by repair synthesis, Nucleic Acids Res., 10, 2065-2084, particular, the purchase of the product does not include or carry any right or license 1982. to use, develop, or otherwise exploit this product commercially and no rights are conveyed to the buyer to use the product or components of the product for purposes 5. Vandeyar, M.A., A simple and rapid method for the including but not limited to provision of services to a third party, generation of selection of oligodeoxynucleotide-directed mutants, commercial databases or clinical diagnostics. This product is sold pursuant to Gene, 65, 129-133, 1988. authorization from Thermo Fisher Scientific and Thermo Fisher Scientific reserves all other rights. For information on purchasing a license for uses other than internal 6. Li, Ch., Evans, R.M., Ligation independed cloning research and development purposes, please contact [email protected] or irrespective of restriction site compatibility, Nucleic Acids Out Licensing, Life Technologies Inc., 5791 Van Allen Way, Carlsbad, California 92008. Res., 25, 4165-4166, 1997. 7. Eun, H.-M. Enzymology Primer for Recombinant DNA Technology, Academic Press. Inc., 1996. PRODUCT USE LIMITATION This product is developed, designed and sold exclusively for research purposes and 8. Tomb, J.F., Barcak, G.J., Regulating the 3'-5' activity of in vitro use only. The product was not tested for use in diagnostics or for drug exonuclease III by varying the sodium chloride development, nor is it suitable for administration to humans or animals. Please refer to www.thermofisher.com for Material Safety Data Sheet of the product. concentration, BioTechniques, 7, 932-933, 1989.